Abstract

The oxidation of naphthalene-1,2-dihydrodiol (ND) too-naphthoquinone (NQ) in the lens is believed to be responsible for the formation of cataracts in naphthalene-fed rats. Studies using either recombinant rat lens (RLAR) or human muscle aldose reductase (HMAR) incubated in vitro with ND in the presence of NAD(P) verified that aldose reductase (EC 1.1.1.21) is the dihydrodiol dehydrogenase that catalyzes the oxidation of ND to NQ. Kinetic studies of Vmax/Kmindicated that RLAR catalyzes the NAD-dependent oxidation of ND with an optimal pH of 9.0. The corresponding activity of HMAR was lower than that of rat enzyme. The metabolite produced by the incubation of RLAR with ND in the presence of 2-mercaptoethanol and NAD in 20 mM phosphate buffer, pH 7.5, was isolated by C18 reversed-phase high-performance liquid chromatography. The elution profile showed the formation of a new peak that was identical with a peak generated when NQ was incubated under the same condition. The metabolite in both peaks was identified as 4-(2-hydroxyethylsulfanyl)-1,2-dihydro-1,2-naphthalenedione (HNQ) by1H and 13C NMR analyses using homonuclear correlation spectroscopy , heteronuclear multiple quantum coherence, and heteronuclear shift correlations via multiple bond connectivities as well as infrared analysis. HNQ is readily autoxidized to 2,3-dihydro-1-oxa-4-thia-9,10-phenanthrenedione. The stoichiometry of 1:1 between the consumption of ND and the formation of NADH for the formation of HNQ implies that rat lens aldose reductase catalyzes a 2e− oxidation of ND to yield the corresponding ketol, which is autoxidized to NQ.

Aldose reductase (EC 1.1.1.21) is a monomeric enzyme with a molecular mass of approximately 35 kDa that utilizes NADPH to reduce a wide range of aromatic and aliphatic aldehydes to their corresponding alcohols. Aldose reductase, along with aldehyde reductase (EC 1.1.1.2) and carbonyl reductases are members of a family of aldo-keto reductases (Turner and Flynn, 1982; Wermuth, 1985). Unlike aldehyde reductase (Mano et al., 1961), aldose reductase predominantly catalyzes the reduction of aldehydes to alcohols. However, recent studies report that aldose reductase can also oxidize some alcohol substrates (Srivastava et al., 1984) and display activity corresponding to that of a dihydrodiol dehydrogenase (Matsuura et al., 1987).

Animals fed naphthalene quickly develop cataracts. This naphthalene-induced cataract has been widely utilized as a model for human senile cataract (Rossa and Pau, 1988). Ingested naphthalene is first oxidized in liver by CYP1A1 to an epoxide that is subsequently hydrolyzed to naphthalene-1,2-dihydrodiol (ND)1by epoxide hydrolase (van Bladeren et al., 1984; Yang et al., 1985;Buckpitt et al., 1987). ND is further metabolized by dihydrodiol dehydrogenase (EC 1.3.1.20) to 1,2-dihydroxynaphthalene, which is eventually autoxidized to 1,2-naphthoquinone (NQ) (Smithgall et al., 1988). Because NQ is highly reactive and quickly forms covalent bonds to various cellular thiols such as glutathaione, cystein, and protein thiols (Smithgall et al., 1988), the formation of NQ in the lens is considered the basic mechanism for the formation of naphthalene-induced cataracts (van Heyningen and Pirie, 1967; van Heyningen, 1979; Xu et al., 1992a). The lens protein modification by naphtoquinone is also considered as a model for lens protein modifications, such as disulfide bond formation, induced by various oxidative insults. Similar protein modifications that are mediated by xanthurenic acid have been reported in senile cataracts (Malina and Martin, 1996).

Interest in naphthalene cataracts has recently been sparked by reports that aldose reductase inhibitors (ARI) can prevent cataract formation in naphthalene-fed rats (Hockwin et al., 1984–1985; Tao et al., 1991;Xu et al., 1992a). Because ARI have been observed to prevent only cataracts induced by ND but not by NQ in lens culture, it has been suggested that ARI prevent naphthalene cataracts by inhibiting the conversion of ND to NQ (Xu et al., 1992b). Based on a previous report (Sato, 1993) that dihydrodiol dehydrogenase is absent in rat lenses and that aldose reductase appears to be the only enzyme in the rat lens that can utilize ND as a substrate, it has been speculated that cataract formation in naphthalene-fed rats resulted from the aldose reductase catalyzed formation of NQ. The present study characterized the metabolites of ND generated by treatment with rat lens aldose reductase and has confirmed that NQ is generated from ND by aldose reductase.

Dehydrogenase Assay of Aldose Reductase.

The reaction mixture (1 ml) contained 1 mM ND, 2 mM NAD, and either 158 pmol of RLAR or 167 pmol of HMAR in either 0.1 M potassium phosphate buffer, pH 7.5 or 0.1 M glycine buffer, pH 9.0. The reaction was initiated by addition of enzyme, and dehydrogenase activity was spectrophotometrically monitored by following the increase of either NADH or NADPH at 340 nm for 4 min with a Shimadzu spectrophotometer (Shimadzu Corp., Kyoto, Japan).

In Vitro Incubation of Aldose Reductase with either ND or NQ.

For high-performance liquid chromatography (HPLC) analysis, the reaction mixture (100 μl) consisted of 131 pmol of recombinant rat lens aldose reductase (RLAR), 7 mM 2-mercaptoethanol, 2 mM NAD, 20 mM phosphate buffer pH 7.5, and 1 mM of either ND or NQ. For the aldose reductase inhibition studies, 10 μM of either the AL1576 or the tolrestat were also included. After the incubation at 25°C for 30 min, the reaction was terminated by addition of 200 μl ice-cold methanol. The mixture was then filtered through a Microcon-3 filter, and the filtrate was analyzed by HPLC.

For liquid chromatography/mass spectrometry (LC/MS) analysis, the incubation was conducted in the same conditions as described above except that 10 mM NAD, 2.5 nmol of RLAR, and either 5 mM ND or 2.5 mM NQ was used.

To investigate the stoichiometry between the oxidation of ND and the consumption of NAD, 400 pmol RLAR was incubated for 3.3 h with 7 mM 2-mercaptoethanol, 1 mM NAD, and 50 μM ND in 20 mM phosphate buffer, pH 7.5. The reaction was terminated by addition of 200 μl of ice-cold methanol, and 5 nmol of oxindole was added as an internal standard. The amounts of ND consumed were determined by HPLC, whereas the amounts of NADH formed were determined by the increase of absorption at 340 nm (ε = 6270 M−cm−1).

Preparation of the 2-Mercaptoethanol Adduct of the Enzymatic Oxidation Product of ND for NMR Analysis.

Ten μmol ND were incubated with 52 nmol of RLAR in a reaction mixture (2 ml) containing 20 μmol of 2-mercaptoethanol and 20 μmol of NAD in 25 mM phosphate buffer, pH 7.5. After incubation at 25°C for 18 h, the metabolites (product I) were extracted with ethyl acetate and purified by preparative silica gel TLC using chloroform/methanol (3:1) as a mobile phase, as described by Smithgall et al. (1988). Product I readily autoxidized, in CHCl3 overnight at room temperature, to a purple-colored product (product II). Product II was purified by preparative silica gel TLC using chloroform/methanol (3:1) as a mobile phase.

Preparation of the 2-Mercaptoethanol Adduct of 1,2-NQ for NMR Analysis.

The 2-mercaptoethanol adduct of NQ was prepared as described bySmithgall, et al. (1988). Briefly, 9.46 ml of 50 mM phosphate buffer pH 7 containing 35 μl of 2-mercaptoethanol and 8 mg of NQ in 0.5 ml of acetonitrile was incubated for 18 h at 25°C. Products were extracted with ethyl acetate and purified as described above.

HPLC.

HPLC was performed using the Pharmacia Smart System (Pharmacia Biotech Inc., Piscataway, NJ) equipped with a μRPC C2/C18 PC 3.2/3 column. The flow rate was 0.25 ml/min with a water/methanol gradient, ramping linearly from 5 to 25% methanol for 10 min, then to 80% methanol at 20 min, with a 5-min hold at 20 min. The eluent was monitored at 245 nm.

LC/MS.

LC/MS experiments were performed on an HP-5989 mass spectrometer (Hewlett-Packard, Palo Alto, CA) interfaced with an HP-1050 HPLC via an HP-59980 particle beam LC/MS, interfaced with an in-line UV detector. The mass spectrometer was scanned from 100 to 300 m/z at 0.8 s/scan. The ion source temperature was 250°C. HPLC separations were performed on an HP ODS Hypersil column (5 μm pore size, 100 × 2.1 mm). A water/methanol gradient was used as described above, at a flow rate of 0.4 ml/min.

NMR Analysis.

Approximately 0.5 mg of the purified samples, dissolved in 120 μl of DMSO-d6, was analyzed on a VXR-500S/Unity spectrometer operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR. Structures of the metabolites were determined using homonuclear correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear shift correlations via multiple bond connectivities (HMBC). The HMBC was set up with a narrow F1 sweep width, a reduction of the number of F1 increments to 256, a change in the long range delay to 6 Hz, and the application of linear prediction to the processing of the final spectrum.

Electron Ionization (EI)-MS.

EI-MS data were collected on a Finnigan TSQ-70 (Finnigan-MAT, San Jose, CA). Introduction was via solid probe. The ion source temperature was 150°C. Full scan data were recorded from m/z 50 to 550 at 0.5 s/scan in electron ionization mode at 70 eV.

Infrared (IR) Analysis.

Results

Effect of pH on the Dehydrogenase Activity of Aldose Reductase.

Effect of pH on dehydrogenase activity of RLAR with ND as substrate was examined in the pH range of 6.5 to 10 (Fig.1). Like many other NAD-dependent dehydrogenases, the dehydrogenase activity of aldose reductase was higher under basic conditions, with a maximum at pH 9. Dehydrogenase activity was observed to be 3- to 5-fold lower at physiological pH.

Twenty millimolar phosphate buffer was used for the pH range of 6.5 to 8.5, while 50 mM glycine buffer was used for pH 9 to 10.

Kinetic Properties of Aldose Reductase with NR as Substrate.

Kinetic analysis of dehydrogenase activity was conducted at pH 7.5 and 9 (Table 1). For RLAR, NAD was a better cofactor than NADP, with Km values for NAD 2- to 4-fold lower than that with NADP. TheVmax values were also higher, with NAD rather than NADP as a cofactor.Vmax/Km values for RLAR, a marker for turnover rate, was 10- to 20-fold higher with NAD than with NADP. Compared with RLAR, dehydrogenase activity of HMAR with ND as substrate was much lower. At pH 7.5, HMAR displayed only trace amounts of dehydrogenase activity with NAD as a cofactor, and at pH 9, the Vmax/Kmof HMAR was less than 10% that of RLAR. Like the rat enzyme, theKm of HMAR with NAD was smaller than with NADP. However, unlike rat enzyme, theVmax/Km of HMAR was slightly higher with NADP than with NAD.

1,2-Naphthoquinone Formation by Aldose Reductase In Vitro.

To investigate whether ND is metabolized to NQ by aldose reductase, ND was incubated with RLAR in the presence of NAD and 2-mercaptoethanol. In the absence of RLAR, the HPLC chromatogram of the incubation mixture displayed a peak corresponding only to unmetabolized ND (Fig.2A). Incubation of aldose reductase with ND clearly resulted in the formation of new metabolite peaks (Fig.2B). The major metabolite peak of ND exactly corresponded to the major metabolite peak obtained when NQ was incubated under identical conditions (Fig. 2C). Addition of the aldose reductase inhibitors, AL1576 and tolrestat into the incubation mixtures significantly reduced the size of these peaks (Fig. 2; D and E).

With this in vitro incubation system, some unidentified peaks at around 3, 4.5, and 15 min were occasionally observed. However, because the appearance of these peaks was not consistent, the identification of these peaks was not pursued in this study.

Stoichiometry between Oxidation of Napthalene-1,2-Dihydrodiol and the Formation of NADH.

To estimate the number of NAD molecules required to oxidize one molecule of ND by RLAR, the consumption of ND and the formation of NADH was quantified after incubation of the enzyme with ND and NAD. The decrease of the substrate, ND, concentration and the increase of NADH concentration were 19.2 ±1.6 and 19.3 ± 0.60 μM (n = 6), respectively, indicating that one molecule of NAD is utilized to oxidize one molecule of ND.

LC/MS.

The major product formed from the enzymatic oxidation of ND was isolated by HPLC and analyzed by particle beam LC/MS (Fig.3). The fragmentation pattern observed for the metabolite of ND was identical with that of NQ. Both spectra contained a molecular ion of m/z 236, corresponding to 4-(2-hydroxyethylsulfanyl)-1,2-naphthalenediol. Loss of the ethanol side chain yielded a fragment ion at m/z 191. Further loss of a hydroxyl group yielded an ion at m/z 174. Loss of the 2-hydroxyethysulfanyl group from the parent ion gave fragment ions atm/z 77 and 159. A fragmentation associated with cleavages between C-1 and C-2 and C-4 and C-4a yielded ions at m/z 105 and 131 at m/z. A cleavage between ring A and B yielded ions at m/z 76 and 160.

LC/MS spectra of the 2-mercaptoethanol adducts of 1,2-naphthoquinone formed either from the oxidation of ND catalyzed by aldose reductase in the presence of 2-mercaptoethanol (A) or from the same reaction with o-naphthoquinone as substrate (B).

Structure of the parent compound (m/z 236) and probable fragment ions are shown at the top.

NMR.

The metabolite of ND formed by treatment with RLAR was analyzed by both 500 MHz 1H NMR and 125 MHz13C NMR (Figs. 4and 5; Table2). Both 1H and13C spectra obtained for the ND metabolite were identical with those of the mercaptoethanol adducts of NQ. The1H NMR spectra showed triplets at 3.27 and 3.75 ppm, corresponding to the methylene protons of the mercaptoethanol substituents at C-9 and C-10, as well as a triplet at 5.19, which are due to a hydroxyl proton at C-10. The aromatic region of each1H NMR spectrum showed the presence of four aromatic protons at 7.67, 7.80, 7.88, and 8.02 ppm, corresponding to protons at C-7, C-6, C-5, and C-8, respectively, as well as a sharp singlet resonance at 6.49 ppm corresponding to an isolated vinyl proton at C-3. The 13C NMR spectrum showed the presence of two dicarbonyls at resonances 175.71 and 178.78 ppm, corresponding to C-2 and C-1. The 13C chemical shifts of the aromatic carbons at C-5, C-6, C-7, C-8, C-8a, and C-4a are 124.9, 135.05, 131.09, 128.24, 130.48, and 133.21, respectively, while the13C chemical shifts of the C-9 and C-10 carbons are 33.98 and 58.32 ppm, respectively. The 13C chemical shift of 157.7 ppm indicated the attachment of an electron withdrawing group, –SR. The site of attachment of the side chain at C-4 was identified by HMBC. The 13C chemical shift of the C-4 carbon (157.71 ppm) was coupled to the proton at C-9, C-5, and C-3, while the 13C chemical shift of the carbon at C-3 was coupled to the proton at C-3 (6.50 ppm). The1H NMR and 13C NMR results supported the hypothesis that the product formed from ND by treatment with aldose reductase and from NQ are both 4-(2-hydroxyethylsulfanyl)-1,2-dihydro-1,2-naphthalenedione (product I). IR spectroscopy further confirmed the presence of carbonyls in product I, and this IR spectrum was identical with that of the 2-mercaptoethanol adduct of NQ (Fig.6). The infrared spectrum of product I displayed a band at 1643 cm−1, which is characteristic of the stretching frequency of a carbonyl of aryl or unsaturated ketones and similar to that (1670 cm−1) reported for NQ (Das et al., 1965). The broad band at 3433 cm−1 reflected the O-H stretching frequency of alcohols, and the band at 1050 cm−1 was consistent with the asymmetric C-C-O stretching vibration of primary alcohols. The band at 3070 cm−1 was consistent with aromatic C-H stretching or olefinic stretching frequencies, and the bands at 2930 cm−1 and 2850 cm−1corresponded to asymmetric and symmetric C-H stretching frequencies.

Fourier transforms IR spectra of the 2-mercaptoethanol adducts formed from either the enzymatic oxidation of ND (A) or from the reaction of NQ (B) in the presence of 2-mercaptoethanol.

The metabolite (product I) from ND was readily autoxidized to form a purple-colored compound (product II). This oxidation product was also analyzed by 1H and 13C NMR (Fig 7; Table3). The 1H NMR spectrum showed the presence of triplets at 3.22 and 4.65 ppm, corresponding to the methylene protons of the mercaptoethanol substituents at C-3 and C-2. Four aromatic protons were present at 7.57, 7.71, 7.75, and 7.90 ppm corresponding to protons at C-7, C-5, C-6, and C-8, respectively. The 13C NMR spectrum showed the presence of the dicarbonyl of the o-quinone as two resonances at 175.34 and 177.34 ppm. Six aromatic carbon chemical shifts were observed between 122 and 136 ppm, and the two methylene carbons at C-2 and C-3 were present at 67.63 and 23.22 ppm, respectively. Carbon 13 chemical shift data indicated that the product was a three-ring system. Evidence for ring closure was based on 1) the13C chemical shifts of the carbons corresponding to C-4a and C-10a at 111.33 and 157.93 ppm, respectively, indicating the attachment of an electron withdrawing group at C-10a; 2) the loss of the C-10a hydrogen and C-2 hydroxyl hydrogen in the1H NMR spectrum of the product; 3) the loss of the C-10a protonated carbon (C-3 in product I) at 120.04 ppm, as indicated by HMQC and the appearance of a new quaternary carbon at C-10a (note that the chemical shift of the C-4b carbon, as well as other carbons, remained unchanged); and 4) the HMBC data showing connectivity between C-2 and C-10a and between C-3 and C-4a.1H NMR and 13C NMR identified the autoxidation product of product I as 2,3-dihydro-1-oxa-4-thia-9,10-phenanthrenedione. Furthermore, the presence of carbonyls in product II was confirmed by IR spectroscopy. The IR spectrum of product II gave a band at 1649 cm−1 (Fig. 8) corresponding to a carbonyl stretching frequency for aryl ketones. The broad O-H stretching band observed at 3433 cm−1in product I diminished to the level of residual H2O. The band at 1092 cm−1was consistent with C-O-C stretching vibration of ethers.

MS.

The metabolite (product I) from ND and its oxidized product (product II) were further analyzed by EI-MS (Fig.9). Because a conversion between carbonyl and aromatic hydroxyls by oxidation/reduction can occur in an EI-MS (Das et al., 1965; Aplin and Pike, 1966), ions at m/z232 and 236, in addition to the molecular ion (m/z 234) of product I, were observed by EI-MS. An ion at 234 was also observed for product II in addition to the molecular ion, m/z 232.

EI mass spectra of the 2-mercaptoethanol adducts (product I) formed from the enzymatic oxidation of ND in the presence of 2-mercaptoethanol (A) and the autoxidation product (product II) of product I (B).

Discussion

Aldose reductase (EC 1.1.1.21) is an NADPH-dependent enzyme that reduces various aldehyde substrates. Aldose reductase also possesses dihydrodiol dehydrogenase (EC 1.3.1.20) activity (Matsuura et al., 1987) and can utilize ND as substrate (Sato, 1993). The present data confirms the identity of the metabolite formed from the oxidation of ND catalyzed by aldose reductase as 1,2-naphthoquinone (NQ).

Evidence supporting the identity of the metabolite of ND by treatment with aldose reductase as 1,2-naphthoquinone (NQ) comes from incubation studies of aldose reductase with ND, NAD, and 2-mercaptoethanol. Incubation resulted in the formation of a new metabolite that was reduced by the presence of aldose reductase inhibitors in the incubation medium. HPLC analysis indicated that this major peak corresponded to the major peak obtained when NQ was substituted for ND in the enzymatic reaction. Also, the LC/MS spectrum of the ND metabolite peak is identical with that of the major peak from NQ.

Catechol formation from dihydrodiols has been detected in the oxidation of 3,5-cyclohexadiene-1,2-diol catalyzed by rat liver dihydrodiol dehydrogenase (Ayengar et al., 1959). However, catechols are extremely unstable in solutions. 4-(2-hydroxyethylsulfanyl)-1,2-naphthalenediol is also readily autoxidized to 4-(2-hydroxyethylsulfanyl)-1,2-dihydro-1,2-naphthalenedione (product I). Although 4-(2-hydroxyethylsulfanyl)-1,2-dihydro-1,2-naphthalenedione is either reduced to the corresponding catechol or further oxidized during EI-MS, only the catechol was observed by particle beam LC/MS. Therefore, it is difficult to distinguish between carbonyls and aromatic hydroxyl groups by MS because of the potential conversion of the two groups by oxidation/reduction (Das et al., 1965; Aplin and Pike, 1966). Further autoxidation of 4-(2-hydroxyethylsulfanyl)-1,2-dihydro-1,2-naphthalenedione was observed to yield 2,3-dihydro-1-oxa-4-thia-9,10-phenanthrenedione, which was also reduced to 4-(2-hydroxyethylsulfanyl)-1,2-dihydroxy-1,2-naphthalene by EI-MS and particle beam LC/MS (data not shown).

The conversions of dihydrodiols to catechols are generally catalyzed by dihydrodiol dehydrogenase. Dihydrodiol dehydrogenase utilizes NADP to catalyze a 2e− oxidation of ND to yield the corresponding ketol, which is autoxidized to NQ (Smithgall et al., 1986). Because aldose reductase utilizes only one molecule of NAD to consume one molecule of ND, aldose reductase, like dihydrodiol dehydrogenase, appears to catalyze the 2e−oxidation of ND to a ketol rather than a 4e− oxidation to directly form NQ. However, RLAR displayed a different preference for cofactor from dihydrodiol dehydrogenase. Although aldose reductase requires NADPH rather than NADH to reduce aldehyde substrates to their corresponding alcohols (Hayman and Kinoshita, 1964; Wermuth, 1985), the oxidation of ND by rat lens aldose reductase is better with NAD than with NADP as cofactor. In contrast to stereospecific dehydrogenase activity of dihydrodiol dehydrogenase, the oxidation of ND by aldose reductase may not be stereospecific. The consumption of more than 50% ND when low concentrations of racemic ND were incubated for prolonged periods (data not shown) suggests that aldose reductase recognizes both theS,S- and R,R-stereoisomers of ND.

The formation of NQ is the key process in naphthalene-induced cataract formation. Because naphthalene dihydrodiol dehydrogenase activity in rat lens is associated with aldose reductase (Sato, 1993), aldose reductase likely produces NQ, the metabolite responsible for naphthalene cataract in rat. Recently, Lee and Chung (1998) have reported that lenses obtained from transgenic mice that overexpress aldose reductase quickly developed cataracts when incubated with ND in vitro. This observation confirms the involvement of aldose reductase in naphthalene cataract formation in rats. The present data confirming the identity of the metabolite of ND formed by treatment with aldose reductase as NQ supports the role of aldose reductase in naphthalene cataract formation.

Although the evidence strongly suggests the importance of aldose reductase in the formation of naphthalene cataract in rats, this must be limited to rats at present. The homologies between the animal and human aldose reductases are generally high (more than 80%). Both rat and human enzymes display the similar activities with the common aldehyde substrates (Sato et al., 1995). However, there are some differences. For example, 17α-hydroxysteroid is a good substrate for both human and bovine aldose reductases, but not for rat enzyme. ND may be one of them. Compared with rat lens enzyme, dehydrogenase activity with ND of human aldose reductase was much lower. Moreover, the human lens contains dihydrodiol dehydrogenase that is lacking in rat lens (Hara et al., 1991). It is unlikely that aldose reductase plays a more dominant role than dihydrodiol dehydrogenase in the formation of catechols from dihydrodiols in the human lens.

Figure 10 summarizes the mechanism for the aldose reductase catalyzed oxidation of ND. Aldose reductase catalyzes a 2e− oxidation of ND to a corresponding ketol, which is subsequently autoxidized to NQ. In the presence of 2-mercaptoethanol, NQ reacts with 2-mercaptoethanol to form 4-(2-hydroxyethylsulfanyl)-1,2-naphthalenediol. This catechol is further autoxidized to the corresponding quinone (product I), which undergoes an intramolecular cyclization to yield another catechol. This catechol is further autoxidized to yield 2,3-tetrahydro-1-oxa-4-thia-9,10-phenanthrenedione (product II), which again may be further autoxidized in the presence of 2-mercaptoehanol.

Formation of the 2-mercaptoethanol NQ adducts initiated by the oxidation of ND catalyzed by aldose reductase.

Whereas catechols are reducing agents, quinones are potent autoxidants. Therefore, the formation of catechols from aromatic dihydrodiols by aldose reductase can lead to the generation of H2O2 and quinones, which may deplete ascorbate and cellular thiols (glutathione, cysteine, and protein thiols) in the lens (Rees and Pirie, 1967; van Heyningen and Pirie, 1967; van Heyningen, 1976). In naphthalene-fed animals, the depletion of ascorbate or reduced form of glutathione also accelerates protein modification by NQ to form naphthalene cataracts.

Acknowledgments

We thank SAIC Frederick (a Division of Science Applications International Corporation, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) for obtaining the NMR, IR, and EI-MS spectra.